Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of embryonic stem cell maintenance and neuronal differentiation[ATAC-seq]

Maintenance of embryonic stem cells (ESCs) and their differentiation to neuronal lineages requires complex genetic regulation of transcription, splicing, and translation, but the interconnections of these processes is not well understood. We found that the splicing regulator polypyrimidine tract binding protein 1 (PTBP1) alters the neuronal transcriptional program. Transcripts for DPF2, a subunit of the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, contain an additional exon (exon 7) in brain. This exon encodes a 14 amino acid insert in the C2H2-type zinc finger domain generating a longer DPF2-L isoform, compared to the canonical short DPF2-S isoform. We find that inclusion of exon 7 in Dpf2 mRNA is strongly inhibited by PTBP1, and loss PTBP1 switches expression from DPF2-S to DPF2-L early in neuronal differentiation. We created CRISPR/Cas9-mediated genome-edited cells that force expression of either DPF2-S or DPF2-L to examine the consequences of this switch in ESCs, in ESC-derived neural progenitor cells (NPCs), and in glutamatergic neurons (GNs). We found substantial changes in gene expression induced by DPF2-L in ESCs and by loss of this isoform in neurons. Chromatin immunoprecipitation in ESCs revealed that DPF2-S but not DPF2-L preferentially bound to genomic regions associated with the stem cell pluripotency transcription factors OCT4, SOX2, and NANOG. In NPC's, DPF2-S preferentially bound to genomic regions co-occupied by the transcription factor NF1, while DPF2-L preferred sites bound by the CTC binding factor, CTCF. The correlation of DPF2 isoform binding with chromatin states identified by ChromHMM revealed that DPF2-S binds to sites enriched in enhancers, whereas DPF2-L-bound sites are enriched for promoters. Altogether, our study identifies a new neuronal BAF isoform that alters its functional targets, and demonstrates how splicing regulatory events during development elicit wide ranging impacts on chromatin organization and transcription. Overall design: To investigate chromatin accessibility across the genome in DPF2-S or DPF2-L expressing cells, we engineered genome-edited mouse ESCs that force express either DPF2-S or DPF2-L. We then performed transposase-accessible chromatin with sequencing assays (ATAC-Seq) in ESCs and ESC-derived NPCs.